The human adenoviruses and herpesviruses are being used as model systems for studying DNA replication in eukaryotic cells. In the past year, we have continued with a genetic analysis of the initiation of adenovirus DNA replication. We have previously shown that the origin of adenovirus DNA replication is comprised of two functionally distinct domains: a ten base pair sequence which probably represents the binding site for a viral initiation protein and an adjacent 20 base pairs which constitutes the binding site for a cellular protein, Nuclear Factor I. We have concentrated in particular on the Nuclear Factor I binding site. Using oligonucleotide mutagenesis we have constructed plasmids with point mutations in the binding site region. Studies with these mutants both in vitro and in vivo have established the following conclusions: 1) the nucleotide sequence specifically recognized by Nuclear Factor I is TGG(N6)GCCAA; 2) the Nuclear Factor I binding site is required for replication in vivo as well as in vitro; 3) tight binding of Nuclear Factor I to the origin is a necessary, but not sufficient, condition for initiation in vitro. We have just begun work with HSV-1. Our efforts to date have concentrated on an attempt to develop a useful in vitro system for studying HSV-1 DNA replication.